The Centers for Disease Control and Prevention (CDC) was recently notified of an individual infected with a monkey cytomegalovirus. Previous reports to CDC of infections with viruses that originated from African green monkeys have largely gone ignored according to Dr. W. John Martin, M.D., Ph.D., founder of the privately funded Centers for Complex Infectious Diseases in Rosemead, California. "Public Health officials have been very resistant, since any airing of this topic will lead to the inevitable conclusion that the Government erred in accepting cytomegalovirus contamination of the African green monkeys used in polio vaccine production." In a 1972 joint government-industry study, cited by Dr. Martin, cell cultures from all 11 monkeys studied were contaminated with simian cytomegalovirus. The scientific community was not informed of this finding. In spite of industry's efforts to curtail contamination, Food and Drug Administration (FDA) officials have confirmed that 3 of 8 licensed polio vaccines released in 1976 have DNA of simian cytomegalovirus. Martin referred to these results in an October 2002 meeting of the Institute of Medicine. His manuscript entitled "DNA Analysis of a Stealth-Adapted Simian Cytomegalovirus" was removed from the published proceedings of the conference.

Martin contends that many of the diseases of epidemic proportions in this county, including autism and attention deficit hyperactivity disorder in children, as well as cancer, chronic fatigue, fibromyalgia and Alzheimer's-like illnesses in adults, are caused by stealth-adapted viruses.

Stealth-adaptation is a process whereby cell-damaging viruses lose components that are normally targeted by the immune system. Some, but not all stealth-adapted viruses are derived from simian cytomegalovirus. Increasingly, Martin is hearing of family illnesses of presumptive infectious origin. He recently submitted a manuscript entitled "Complex multi-system illnesses occurring within a family: Presumptive evidence for an infectious disease process" to the CDC Journal of Emerging Infectious Diseases, but its publication was declined. "It is hard to wake them up" says Martin "and near impossible to break through the self protective barrier of those who recognize that cytomegalovirus contaminated polio vaccines used in Central Africa could have triggered the formation of the AIDS virus. CDC has a collection of sera from polio vaccine immunized African children that, under better leadership, would have been tested for cytomegalovirus antibodies." Except for legal purposes, Martin has been prohibited from communicating results of stealth virus testing to individuals with complex unexplained illnesses. A petition recently sent to CDC requesting that it tests autistic children for stealth-adapted viruses was seemingly futile.

"The real tragedy of CDC" says Martin "is the public's expectation that it will aggressively pursue every indication of potentially infectious agents contributing to human diseases. Doing nothing is tantamount to allowing disease to go unchecked. One way to establish dialogue with the CDC is through the litigation process. Alternatively, a Congressman or Senator willing to risk the ire of the pharmaceutical industry must insist that CDC immediately address the issue of cytomegalovirus contamination of polio virus vaccines and interview some of the many patients infected with stealth-adapted viruses.

Dr. Martin says, "Now is the time for well designed clinical studies to evaluate methods to prevent disease transmission and to help restore normal brain function to stealth-adapted virus infected individuals. The public needs to be informed and our Government agencies need to respond."

More information, including copies of the manuscripts submitted to the Institute of Medicine and CDC Journal of Emerging Infectious Diseases, is available at www.s3support.com
# # #

Sunday, October 10, 2004

Detection of Stealth Viruses in Human Tissues

W. John Martin, M.D., Ph.D.

Presentation made at a meeting entitled:
"Consultation on Detection of Simian Cytomegaloviruses in Human Tissues"
Sponsored by the National Institute of Allergy and Infectious Diseases (NIAID) and held in Room 1A1 Solar Building, 6003 Executive Blvd. Rockville MD 20892, July 1, 1996

The purpose of the meeting is to "evaluate existing data and to discuss approaches for the detection of Simian CMV in human tissues." This issue arose from a presentation made to the Institute of Medicine Vaccine Safety Forum on November 6, 1995 and from reports of stealth viruses infecting patients with neuropsychiatric illnesses. Published data indicate that some stealth viruses have arisen from African green monkey simian cytomegalovirus (SCMV), a potential contaminant of polio vaccines. Requests for immediate FDA/CDC action and for financial support to continue these studies have been denied.

INTRODUCTION
As reflected in the following quotations, vaccine contamination has been a recurring concern with all live vaccines, including polio.
"The use of living organisms for immunization on a large scale is always liable to occasional disasters. It may be possible to be quite certain that the germ we intend to inoculate is harmless, but there is always a possibility of some unwanted and harmful micro-organism finding its way into the material. Cultures or tissue extracts containing living organisms to be used for inoculation cannot be sterilized, so there is no automatic method for which their safety can be assured. In any reputable laboratory every safeguard will be taken to eliminate any possibility of danger from contamination, but all these precautions depend ultimately on human vigilance, which is never infallible."
F. MacFarlane Burnet, 1940 "As monkey kidney tissue is host to innumerable simian viruses, the number found varying in relation to the amount of work expended to find them, the problem presented to the manufacturer is considerable, if not insuperable. As our technical methods improve, we may find fewer and fewer lots of vaccine which can be called free from simian virus."
Hilary Koprowski, communication to Congress, 1961 "If a vaccine program were to be initiated today, one would surely not import wild monkeys from Africa, create short term primary kidney cultures, add a human virus and administer the crude gamish derived from the virally infected cells to virtually every child in the country. Nor would one want to withhold applying the many molecular biological techniques developed over the last 30 years to assess vaccine purity. Yet this is essentially the situation with live polio vaccine and comparable arguments can be made for other human and animal viral vaccines."

W. John Martin, California Dept. of Health sponsored meeting on 20th Century Plagues, March 1, 1996 EVIDENCE OF POLIO VACCINE CONTAMINATION
During the early period of polio vaccine production, greater than half of the rhesus monkey kidney cultures established for polio vaccine production were discarded due to the presence of adventitious viral agents.
In 1959, NIH researcher, Dr. Bernice Eddy went beyond the approved testing protocols and began to test whether rhesus monkey kidney cultures contained a virus able to induce tumors in hamsters. Her positive results were suppressed. Proof of the oncogenicity of a virus frequently present in rhesus monkey kidney cells was subsequently provided in 1960 by Drs. Sweet and Hilleman of Merck. The virus was designated SV-40, since it was the fortieth virus so far found in rhesus monkeys. SV-40 was shown to contaminate many lots of polio and adenoviral vaccines. It was not completely inactivated by the 0.4% formaldehyde used to produce Salk vaccine. There was no recall of the already distributed contaminated lots of polio vaccines. Instead, SV-40 neutralized polio vaccine was simply transferred to African green monkey kidney cultures for the continued production of vaccine.
Between 1961 and 1963, the United States also switched from formaldehyde treated Salk vaccine to Sabin's live attenuated polio vaccine. Again, there is documentation of the willingness of those in-charge of the vaccine program to "turn a blind eye" to the possible presence of contaminating monkey viruses:
"Manufacturing regulations limited the observation of tissue culture control bottles (containing 25% of the monkey tissue used to manufacture polio vaccine) to 14 days - a time period chosen for the specific purpose of passing vaccine lots made in tissue harboring extraneous viruses in 'eclipse'. Longer observation periods (21 or 28 days) were rejected because the expected appearance of contaminants might require rejection of a monopool of the vaccine. The NIH adopted the 14 day time period and manufacturers switched to the untested African green monkey kidney tissue in which SV-40 was not indigenous. Everyone at the meeting agreed that the potential for the presence of a then undetectable virus in African green monkey tissue was great, but since nothing could be detected, the material would pass regulations for production as drawn."
NIH Meeting, circa 1961 "I told Dr. Murray that there was some concern at Lederle about a possible requirement barring the use of African green monkey kidney as the substrate for the growth of attenuated polioviruses...Dr. Murray had stated that the adventitious agents that Dr. Kendall Smith is presumably detecting by his techniques are of little consequence for an oral preparation in that such a large experience exists with the use of oral polio vaccine without any evidence of trouble relating to these agents."
Lederle memo August 23, 1968 "All eleven monkeys studied demonstrated the presence of CMV-like agents. These monkeys all originated from Kenya over a short period of time. Seven of these monkeys would have passed our existing test standards, only one of these monkeys would have passed our test methods using Lederle 130 human diploid cell strain..... We plan to continue to process monkeys at the rate of five per week, probably through October 1972, to provide us with thirty million doses of trivalent poliovirus vaccine...... Unless and until Pfizer's Diplovax is in abundant supply, the BB [Bureau of Biologics] cannot risk Lederle being off the market."
Lederle's "Cytomegalovirus Contingency Plan", 8/4/1972 "I do not believe our problem with the slow release of specific lots of Orimune(R) are a result of a Pfizar influence..... Furthermore, if the Bureau wanted to restrict us they could bring up the subject of CMV (Cytomegalovirus) in our substrate (i.e. African green monkey kidney tissue) which they have not done, even though they have told us the monkeys in the collaborative study performed in 1972 were all positive for this agent."
Memo to the President of American Cyanamid, 1973 An atypical cytomegalovirus, subsequently shown to be an African green monkey simian cytomegalovirus (SCMV), was apparently cultured from a brain biopsy of a 7 year old boy with an atypical encephalopathy. The virus became known as Colburn SCMV.
Dr. Alford and colleagues, 1973 "According to Dr. Petricciani of the BoB, monopool PP3-444 contains 'reverse transcriptase' an enzyme often found to be associated with a certain class of tumor viruses."
American Cyanamid memo, October 1976 "The same agent which is present in 444 giving rA.dT activity can be transferred to FCS-7, MRC-5 and FRhL-1 cells."
Memo from J. Petricciani, 1976 "To our dismay, purified virus RNA was always contaminated by large quantities of unlabeled cellular DNA..... If the presence of cellular DNA in vaccine preparations is a routine occurrence, it would seem possible that there would be a possibility of occasional transfer of genetic information of the viral host species to the vaccine recipient species."
John Dahlberg's review of EM of polio batch PP3-444 January 1977 Not all of the DNA in polio vaccines is from normal monkey cells or from the fetal calf serum used to grow the monkey cells.
Results of G. Aulakh and W. John Martin in 1977 "Stop worrying about it, every time you eat an apple you ingest foreign DNA."
Comments from BoB Director to W. John Martin, 1977 A recipient of an experimental rubella vaccine made in African green monkeys is considered to have been infected with a simian cytomegalovirus.
As remembered by a CDC technician (J.P.) Significant reverse transcriptase (RT)-like activity is present in 3 of 12 batches of polio vaccine. Six of the 12 batches appear to induced high levels of RT-like activity in cultures of human peripheral blood lymphocytes.
Handwritten notes of a BoB studies dated Aug-Oct, 1985 SV-40 virus is detected in human mesotheliomas, ependymomas, choroid plexus brain tumors and osteogenic sarcomas.
Results from Drs. Carbone, Lednicky and Butel, 1992-1996 DNA sequencing of a virus repeatedly isolated from a patient with chronic fatigue syndrome (CFS) shows that it originated from African green monkey simian cytomegalovirus (SCMV). A molecularly related "stealth" virus is also isolated from a patient with acute encephalopathy and a 4 year history of bi-polar psychosis.
Results of W. John Martin 1991-1995 SUMMARY OF EXPERIMENTS LEADING TO DETECTION OF STEALTH VIRUSES
The project was initiated in 1988 to see if patients diagnosed as having CFS were virally infected. Using the polymerase chain reaction (PCR), a primer set reactive with all human herpesviruses, gave weak positive signals with blood from some CFS patients, but not with blood of laboratory control personnel. CFS patients also not infrequently, gave a low level response using primers designed to amplify the tax gene of human T lymphocytotropic viruses.
The potential significance of the marginal responses seen in CFS patients became clearer in 1990 when strikingly positive PCR responses occurred with cerebrospinal fluid (CSF) samples from two patients and on a brain biopsy of a third patient. All three patients had unexplained severe encephalitis-like illnesses. The brain biopsy came from a 39 year old school teacher who had gradually lost her capacity for written and oral communication, but was otherwise alert with no localizing neurological signs. She did, however, have an abnormal MRI with periventricular lesions. The brain biopsy showed minimal cellular changes with no inflammation. Complete and incomplete forms of herpesvirus-like particles were present in several of the glial cells examined by electron microscopy. Some of the virus positive cells showed marked vacuolated changes and lipid accumulation. This patient has since further deteriorated and is in a vegetative state. An MRI performed in March 1996, shows severe atrophy with marked ventricular dilatation. The PCR positive CSF samples tested in 1990, were both acellular, indicating an absence of inflammation. One came from a premature infant with delayed neural development. This child gradually improved but still performs at a below average level. His mother has had an MRI for headaches and other symptoms which have developed over the last several years. The other CSF sample came from an adolescent with residual severe brain dysfunction three years after what was labeled an atypical herpes simplex encephalitis. His younger brother also had an unexplained encephalitis-like illness with coma from which he had recovered.
I had made earlier attempts to grow human herpes virus-6 (HHV-6) from CFS patients using cord blood lymphocytes. Routine human fibroblast cultures from CFS patients were also established. The fibroblasts frequently showed a delayed, transient cytopathic effect (CPE) somewhat suggestive of human CMV. Buoyed by the PCR findings in the severe encephalopathy cases, attempts were again made to culture a virus from a PCR positive patient. I became aware of a 43 year old healthcare worker who had not fully recovered from an unexplained subacute encephalitis-like illness in July of 1990. Her blood tested positive using both the HTLV and the herpesvirus primer sets. An additional blood sample was collected for culture. After 6 weeks, enlarged, rounded, vacuolated cell syncytia appeared in both human and monkey derived cultures. The CPE could be passed to many other cell types and to cells of multiple species. Electron microscopy showed numerous complete and incomplete herpesvirus-like viral particles. The foamy, vacuolated cells did not, however, stain with antisera specific for the immediate-early gene of human CMV. Nor did the cells stain with antisera specific for HSV, HHV-6/7, VZV, adenoviruses or enteroviruses. PCR assay using the HTLV tax gene primer gave two well defined products. Sequencing of one of the PCR products (GenBank accession # U09212) identified a region of partial sequence homology to the UL34 gene of human CMV. The sequence of the other PCR product (GenBank accession # U09213) was suggestive of a possible herpesvirus origin but could not be aligned with any of the known herpesviruses. Viral DNA was isolated and cloned. A region of sequence homology to African green monkey simian cytomegalovirus (SCMV) was noted in early 1994 and reported to CDC. Dr. Gary Hayward of Johns Hopkins University submitted additional sequence data on SCMV to GenBank in December 1994. From his sequence data, it was unequivocal that the CFS patient's virus had originated from SCMV. This conclusion was reported to FDA in March of 1995.
In 1991, I coined the term "stealth" to describe the viruses that were being identified in blood and CSF cultures, and which had been seen on electron microscopy of the brain biopsy. This term was used primarily because it denoted a cytopathic infection occurring in the absence of inflammation. The SCMV-derived virus isolated from patient D.W., was designated stealth virus-1. She has remained quite disabled with fatigue and cognitive impairment. Positive cultures are still obtainable from this patient but complete viruses are no longer seen by electron microscopy and no longer react in PCR assays using HTLV primers.
A molecularly related virus (stealth virus-2) was isolated in February 1991 from the CSF of a patient with an acute severe encephalopathy. The 23 year old female patient (B.H.) had a 4 year history of a manic depressive illness. Her encephalopathy was attributed to an attempted suicide from drug overdose, for which no evidence was subsequently found. A diagnosis of viral infection was excluded mainly because of the paucity of cells in her CSF and the lack of a response to acyclovir therapy. The patient has remained in a vegetative state since 1991. DNA sequencing of a PCR amplified product from her infected cultures showed that it was similar, but not identical, to the SCMV-derived stealth virus-1. Numerous other stealth viral isolates have been obtained from patients with various types of illnesses and from some apparently normal controls. I am awaiting resources to perform a true prevalence study as well as sequencing analyses on several of the isolates.
Restriction enzyme digests of stealth virus-1 have been cloned and partially sequenced. The genome appears to comprise multiple fragments of approximately 20 kilobase pairs. The virus displays an unusually high degree of sequence microheterogeneity suggesting infidelity of DNA replication or an alternative replication strategy. The concept of a fragmented, unstable viral genome is consistent with electron microscopic observations on several additional stealth viral isolates. It has an important implication for the limited utility of molecular techniques to screen for these viruses. While replication errors may help explain recovery from infection in the absence of an inflammatory response, it does raise some concern about the potential oncogenicity of stealth viruses.
Stealth virus-1 induces an acute neurological illness when inoculated into cats. The early manifestations developed within a week and included gingivitis, bloody ocular and nasal discharge, lymphadenopathy, pupil dilatation with photophobia (squinting in response to light) and nuchal hair loss from rubbing against the cage. There was a reduction in body temperature beginning in the second week which averaged 0.6oF at 2 weeks and 0.8oF at weeks 3 and 4. Most striking was the marked behavioral changes in all of the virus-inoculated cats. They lost the playfulness that was present prior to injection and became reclusive and irritable. They resisted being handled and the animal caretakers resorted to wearing leather gloves. By palpation, the enlarged lymph nodes and various muscle groups were identified as being painful for the animals. The severity of the illness peaked at around 4 weeks with definite improvement noted in the cat necropsied at 6 weeks. A cat that was maintained to 15 weeks had resumed normal activities by week 10 and appeared to be symptom-free. Histological examination of animals' brain tissue showed foci of cells with cytoplasmic vacuolization and an absence of any inflammatory reaction. Electron microscopy confirmed the presence of occasional herpes-like viral particles along with accumulations of viral-like granular and membranous structures suggestive of subgenomic viral expression. A cat injected with heated stealth virus-1 did not become sick upon receiving unheated stealth virus-1, but did succumb to neurological illness when subsequently injected with the stealth virus isolated from another patient.
Unpublished surveys suggests that the pets of patients with CFS have a higher than expected incidence of neurological illness. This observation is consistent with a human to animal disease transmission. Furthermore, blood from a symptomatic cat transmitted disease to a healthy recipient cat. The brains of both animals showed vacuolating cellular changes characteristic of stealth viral infection.
STEALTH VIRUSES ISOLATED FROM PATIENTS WITH SEVERE ENCEPHALOPATHY
Since beginning these studies, many patients have been identified with complex neuropsychiatric and rheumatic illnesses that exceeded the normal bounds of existing medical specialities. The finding of a positive stealth viral culture in such patients may help unravel the true cause of their illness and provide a fresh approach to therapy and disease prevention. Children with autism and other pervasive developmental disorders have also shown consistent evidence for stealth viral infection. Clinical observations and interviews with many of the neurologically affected patients, lend support to the concept of a spectrum of potentially infectious neuropsychiatric illnesses. The distinction between patients labeled as having different illnesses may simply reflect infection of different regions of the brain and varying psychological reactions to the resulting dysfunctions. Support for a viral encephalopathy etiology in some of these patients has been provided by detailed histologic examinations of additional brain biopsies which reveal similar cytoplasmic and nuclear changes as those seen in the brains of infected cats. In one of the biopsies, there was an additional vasculitis component, while in another, there was an overall low level inflammatory reaction. Electron microscopy readily confirms the cellular vacuolization, often accompanied by mitochondrial disruption. Although intact viruses may be rare, prominent accumulations of viral-like materials can usually be seen.
A promising clinical aspect of these infections is the marked improvement that has occurred in some of the severely ill patients. A better understanding of the mechanisms involved in their recovery process offers some hope for the many patients in whom their tragic disease simply persists for years. Detailed clinical summaries of twelve such patients, including several healthcare providers who may have been occupationally infected, was provided to the FDA and to the CDC in June 1995.
GOVERNMENTAL RESPONSE TO REPORTS OF ATYPICAL VIRAL INFECTIONS
No specific enquiries were received about any of the patients reported to the FDA and CDC. Nor was the requested financial support provided to study the prevalence of SCMV-derived stealth virus infection in humans or to perform sequencing on additional viral isolates. FDA has sided with American Cyanamid to legally resist making the polio vaccine lots administered to a child who has an unexplained neurological disease, (W.W.), available for independent analysis for contaminating viruses. FDA and CDC appear reluctant to openly address whether vaccines could have contributed to the increasing incidence of dysfunctional brain syndromes, including CFS. The possibility that monkey viruses may have entered into the nation's blood supply certainly warrants the attention of FDA.
The Gulf War syndrome has stricken many American servicemen, but has largely been played down as a possible infectious disease. Considering all of the counterproliferation efforts to combat biological terrorism, the Military should be concerned with the quality of the vaccines and of blood products, including gamma globulin preparations, given to its troops.
Most individuals are unaware of the enormity of the world's trading in vaccines and blood products. As in other large industries, the public needs reassurance that the agencies designed to act as watch-dogs do not simply become shields for questionable business practices. Legislation allowing the bypassing of FDA regulations of blood products exported from this Country, including testing for HIV, undoubtedly contributed to the spread of this infection throughout the world. It would be a sad commentary, if the grand contributions of Dr. Salk and Dr. Sabin to the eventual elimination of polio were to be marred by inattention to problems not fully appreciated at the time of their discoveries.
RECOMMENDATIONS
Immediate scientific disclosure of non-published studies relating to the safety of polio vaccines conducted by FDA and/or by American Cyanamid (Lederle).
Independent (non-Governmental and non-industry) scientific review of the production, source material and testing protocols utilized in the currently licensed live and inactivated polio vaccines.
A statement to the public that until these data have been reviewed and recommendations implemented, there should be no compulsory polio vaccination.
Testing of blood products, including gamma globulin preparations for stealth viruses and for SV-40.
Prevalence study on stealth and SV-40 viral infections in the general population and in various disease groups.
Notification through the MMWR of the need to report cases of atypical encephalopathic illnesses.
A waiver given along with any product submitted for IND or NDA approval giving the FDA the authority, and indeed the obligation, to inform the medical and scientific communities of any information it gathers related to the product's safety.
REFERENCES
1. Martin W.J. Stealth viruses as neuropathogens. CAP Today 8 67-70, 1994.
2. Martin WJ, Zeng LC, Ahmed K, Roy M. Cytomegalovirus-related sequences in an atypical cytopathic virus repeatedly isolated from a patient with the chronic fatigue syndrome. Am J Path. 145: 441-452, 1994.
3. Martin WJ. Stealth virus isolated from an autistic child. J Aut Dev Dis. 25:223-224, 1995.
4. Martin WJ, Ahmed KN, Zeng LC, Olsen J-C, Seward JG, Seehrai JS. African green monkey origin of the atypical cytopathic 'stealth virus' isolated from a patient with chronic fatigue syndrome. Clin Diag Virol. 4: 93-103, 1995.
5. Cristaudo A, et al. Molecular biology studies on mesothelioma tumor samples: preliminary data on H-ras, p21 and SV40. J. Environ Path 14: 29-34, 1995.
6. Lednicky JA et al. Natural simian virus strains are present in human choroid plexus and ependymoma tumors. Virology 212: 710-717, 1995.
7. Carbone M, Rizzo P. Procopia A., et al. SV40-like sequences in human bone tumors. Oncogene (in press).
8. Martin WJ. Severe stealth virus encephalopathy following chronic fatigue syndrome-like illness: Clinical and histopathological features Pathobiology July 1996
9. Martin WJ, Glass RT. Acute encephalopathy induced in cats with a stealth virus isolated from a patient with chronic fatigue syndrome. Pathobiology 63: 115-118, 1995.
10. Gollard RP, Mayr A, Rice DA, Martin WJ. Herpesvirus-related sequences in salivary gland tumors. J Exp Clin Can Res.15: 1-4, 1996.
11. Martin WJ. Genetic instability and fragmentation of a stealth viral genome. Pathobiology July 1996.
12. Martin WJ. Simian cytomegalovirus-related stealth virus isolated from the CSF of a patient with bi-polar psychosis and acute encephalopathy. Pathobiology (in press).
13. Martin WJ. Stealth viral encephalopathy: Report of a fatal case complicated by cerebral vasculitis. Pathobiology (in press).
14. Information can also be found on the World Wide Web at URL http://www.ccid.org Read the CDC's Response here

Stealth-adaptation is a mechanism that allows cytopathic viruses to evade immune elimination through the deletion of genes coding the major antigens targeted by the cellular immune system. A prototype stealth-adapted virus cultured from a patient with chronic fatigue syndrome (CFS) was readily transmissible to cats in which it induced an acute encephalopathy without localizing neurological signs. Vacuolating cellular damage was observed in many tissues of the animals, including the brain, in the absence of an accompanying inflammatory response. The cultured virus was cloned and partially sequenced. It comprises a fragmented, genetically unstable, genome. It has viral sequences that can be aligned to various regions of the genome of human cytomegalovirus (HCMV). Where the comparison can be made, the sequences match much more closely to those of African green monkey simian cytomegalovirus (SCMV), indicating an unequivocal origin from SCMV. In addition to SCMV-derived sequences, the cytopathic virus has acquired both cellular and bacteria-derived DNA. Cellular sequences include putative oncogenes and sequences related to those of endogenous retroviruses. Many of the bacterial sequences match closely to known genes of Brucella, while other genes show greater homology either to mycoplasma or to Streptococcus. The presence of bacteria-derived sequences has led to the secondary designation of this type of novel microorganism as viteria. Molecularly heterogeneous viruses, inducing similar characteristic cytopathic effects in culture (and when examined, non-inflammatory vacuolating cellular damage in brain and other tissues), have been cultured from numerous patients with severe neurological, psychiatric, immunological and neoplastic diseases. The differing clinical manifestations in infected patients may reflect the assimilation of different cellular and other sequences in various stealth-adapted viruses. Tissue culture provides a valuable screening method for the detection of stealth-adapted viruses and for characterizing novel virus-associated products. Cultures can also help avoid the misidentification of stealth viruses for conventional viral and bacterial pathogens purportedly associated with chronic diseases.

Introduction

Chronic illnesses are typically categorized according to the predominating clinical manifestation. Accordingly, they come under the purview of different types of medical specialists, such as psychiatrists, neurologists, rheumatologists, endocrinologists, cardiologists, gastroenterologists, oncologists, etc. While this specialization fosters expertise in diagnosis and therapy, it tends to deemphasize the overlapping clinical features shared by many chronic debilitating illnesses. Symptoms such as fatigue, insomnia, impaired mood and cognition, widespread aches and pains, etc., typically occur in most chronically ill patients regardless of the underlying diagnosis. Individual patients will not uncommonly qualify for having two or more chronic diseases while family histories often reveal a constellation of medical problems among different family members. While most acute infections tend to give rise to a consistent clinical pattern of illness, this is not the general rule for chronic viral or bacterial infections. It is conceivable, therefore, that a common chronic infectious process could be involved in the currently widely recognized increases in many types of chronic illnesses. This consideration does not exclude important additive contributions of genetics, psychosocial, other environmental factors, autoimmunity, etc., in individual patients leading to their particular complex disease process. To help stem the ever increasing prevalence of chronic illnesses, it is vitally important not to overlook an infectious and, therefore, potentially contagious component. This paper reviews evidence that implicates chronic non-inflammatory, stealth-adapted viruses in many forms of human and animal diseases.

Stealth-adaptation refers to the ability of a conventional cytopathic (cell damaging) virus to lose portions of its viral genome that includes genes coding for components normally recognized by the cellular immune system. Although not widely appreciated among most virologists, only a relatively few number of virus coded components provide the target antigens for the vast majority of anti-viral cytotoxic T lymphocytes (CTL). Deletion of these few select genes can essentially enable a virus to avoid effective immune recognition. The best studied stealth-adapted virus is a derivative of an African green monkey simian cytomegalovirus (SCMV). This virus presumably arose from an SCMV contaminated vaccine, such as a live polio virus vaccine. These vaccines were routinely produced in fresh kidney cell cultures obtained from African green monkeys. In a 1972 joint Lederle-Bureau of Biologics cooperative CMV study 1 , all eleven monkeys studied demonstrated the presence of CMV-like agents. Seven of the monkeys would have passed [Lederle's] existing test standards; only one of these monkeys would have passed test methods using Lederle's 130 human diploid cell strain. Lederle proposed that it would begin treating monkeys with DNA antagonists during their isolation period, then remove the kidneys and test exhaustively for the presence of CMV in the kidney tissue. Even assuming that this was done, it is disturbing that 3 of 8 tested lots of live polio virus vaccine released later in the 1970's were recently confirmed as containing SCMV DNA 2 . The present paper will provide a brief summary of studies leading to the isolation and subsequent molecular characterization of a stealth-adapted SCMV-derived virus.

Stealth Virus Detection and Isolation

The research program began in 1988 as a search for a viral cause of the chronic fatigue syndrome (CFS). It was based on the recent description of human herpesvirus-6 (HHV6), and on the availability of a highly sensitive molecular diagnostic assay, known as the polymerase chain reaction (PCR). 3 The PCR assay comprises reacting a sample with relatively short pieces of synthesized DNA (primers) that selectively bind to the flanking sequences of a relatively small section of double stranded DNA present in the particular sample being tested. The DNA is subjected to repeated rounds of heating to separate the DNA strands, cooling to allow the primers to bind and to be extended by DNA synthesis mediated by a heat resistant DNA polymerase enzyme. This process leads to exponential amplification of the targeted section of DNA, which greatly facilitates its detection and sequencing. Although PCR assays can be exquisitely sensitive, they can yield misleading information. For example, if a pathogen has a mutation or deletion in the targeted section of DNA, a negative PCR assay can result even though much of the remaining parts of the microorganism may be present. Conversely, if the primers are directed against sequences that are partially shared by different microorganisms, spurious identifications can be made based on a positive PCR. In spite of these limitations, PCR assays have proven extremely useful in the search for unexpected pathogens, including as detailed below the detection of stealth-adapted viruses.

The initial PCR assay employed was based on limited known DNA sequences of HHV6. The assay generally yielded negative results in patients with CFS. The PCR assay was modified so as to be cross-reactive with other known human herpesviruses. Other PCR primer sets were also designed to detect various retroviruses. The PCR assays were shown to be even broadly cross-reactive than anticipated, with for example, amplification occurring with adenoviruses by the herpesvirus-reactive primers. The assays were applied to blood samples of CFS patients. Depending on the conditions used, positive responses were seen with approximately a third to a half of the patients tested. 3-5 Parallel testing of blood samples from the vast majority of apparently healthy individuals gave negative results. Some of the positive patient responses were quite striking in their intensity. The importance of the work became apparent when very strong PCR reactivity was seen using a cerebrospinal fluid (CSF) sample from both a newborn child with hepatosplenomegaly, seizures, and impaired neurological development. This finding was followed shortly by a similar strong reactivity using CSF obtained from an adolescent with major neurological damage resulting from what had been considered a missed diagnosis of herpes simplex encephalitis. In both cases the CSF was totally devoid of any cellular inflammatory reaction and routine virus culture assays were reported as being negative. A positive PCR was also seen using a portion of a stereotactic brain biopsy performed on a school teacher with periventricular lesions identified using magnetic resonance imaging (MRI). 4 Again, the striking feature of the brain biopsy was the lack of any inflammatory reaction. The brain cells did, however, show lipid filled vacuoles, damaged mitochondria and accumulations of irregularly shaped pigmented inclusions. These observations were consistent with non-inflammation-inducing cell damaging (cytopathic) viruses causing a spectrum of neurological illnesses. The term “stealth” was introduced to convey the apparent ability of the viruses to avoid effective immune recognition that would trigger an anti-viral inflammatory reaction. The task was to isolate such a “stealth-adapted” virus.

Many types of viruses can be detected by observing for a cytopathic effect (CPE) on cells grown in tissue culture. Using standard viral culture techniques suitable for the detection of human cytomegalovirus (HCMV), highly suggestive, but non-persisting cellular damage had previously been observed in cultures performed on numerous CFS patients. Clearly positive PCR assays were obtained on blood samples of a patient who experienced an encephalitis/meningitis-like illness in July 1991. A determined effort was made to culture her blood. After some 6 weeks delay, a strong sustainable cytopathic effect was observed. 6 This individual has remained cognitively impaired with personality changes and marked fatigue. She has remained on disability with a diagnosis of CFS.

The striking tissue culture finding was the formation of foamy vacuolated cells that were comparable to what were seen in the brain biopsy. Repeated cultures from this patient provided similar positive results with the additional observation that the CPE would develop much earlier if the culture medium was frequently (daily or every 2nd day) replaced with fresh medium. The positive culture did not react in standard serological typing assays using antibodies specific for conventional human herpesviruses (CMV, HHV6, Varicella zoster, Herpes simplex or Epstein Barr), nor for enteroviruses or adenoviruses. It was also negative for human CMV and HHV-6 using specific PCR-based assays. The patient-derived cytopathic virus was successfully cultured in multiple cell lines from human and animal sources 6 and could even replicate in insect-derived cells. Virus aliquots were submitted to the American Type Culture Collection (ATCC) for long term storage and public access (Accession number VR 2343).

Soon, thereafter, a slightly differing appearing, but otherwise very comparable, strongly positive culture was obtained in a physically different laboratory. The CPE developed in human fibroblasts three weeks after being inoculated with a CSF sample from a patient with a 4 year history of a bipolar, manic depressive illness. The woman had deteriorated clinically and developed seizures prior to admission to L.A. County Hospital . An aliquot of the second sample was sent to the Los Angeles County Public Health Laboratory. Following confirmatory culturing, the County laboratory reportedly sent a sample to the California State Laboratory. This virus was not identified by either the County or State laboratory and was dismissed as a probable contaminant. Electron microscopy on both of the patients' cultures confirmed the presence of numerous herpesvirus-like particles.

Many additional blood and occasional CSF samples were found to induce a foamy vacuolating CPE in viral cultures on both human fibroblasts and rhesus monkey kidney cells. Adjustments to the culturing techniques, especially the use of serum free medium, frequent replacement of the medium, and freeze-thawing of the cells prior to culturing, led to the more rapid development and greater intensity of the CPE. The basic feature of positive cultures was the transformation of the normally thin, spindle-shaped fibroblasts into swollen, rounded, vacuolated, foamy (fat-filled) cells, with a tendency to form clusters. The extent of cell swelling, size of cell clusters, formation of syncytia from cells fusing with one another, amount of fat accumulation both in the cells and in the medium, and the extent and appearance of accompanying accumulations of fine to coarse pigments, all varied more or less independently between the cultures obtained from different individuals. Consistent positive/negative readings, as well as more detailed descriptions of the positive cultures were obtained by impartial observers and the distinction between patient and control populations confirmed in several double-blinded studies. The positive patients had a wide variety of neurological, neuropsychiatric and auto-immune diagnoses. 7-10 Families were identified with various diagnoses among the different family members, but with very similar cytopathic changes seen in cultures.

Animal Studies

Cells from the virus culture of the initial culture positive CFS patient were inoculated into cats. The cats experienced profound behavioral changes. 11 Within a week they transformed from friendly, frisky happy-go-lucky favorites of the University animal facility, to frightened, reclusive animals shying away from the light. Several of the cats developed bald areas of skin upon their heads and necks from rubbing against the cage. Some had exudates from their nose from scratching. Gentle handling of the animals revealed painful muscles and enlarged lymph nodes. The acute illness peaked at 1-2 weeks with significant clinical recovery seen in the animals maintained from 5-16 weeks post inoculation. Necropsy showed widespread non-inflammatory vacuolating cellular changes. Heat killed virus did not induce disease, and in fact, provided protection against subsequent inoculations of the same virus. The animal became ill when inoculated with a virus isolated from a different patient who had been diagnosed with systemic lupus erythematosus. Animals of patients were also reported to show various symptoms, and yielded positive cultures when blood samples were tested. Blood samples from ill cats were inoculated into healthy cats that subsequently became ill and virus culture positive.

Sequencing Studies

The primer sets that gave positive responses PCR reactions in the blood of the virus culture positive patient with CFS, also yielded strong reactions when tested on each of several repeat positive cultures from this patient. Two PCR products of approximately 1.5 x 10 3 nucleotides were isolated and sequenced. 6 One product (GenBank accession number U09212) contained a sequence that showed a statistically significant homology to a region of the genome of HCMV corresponding to the UL34 gene (UL refers to the Unique Long segment of the human CMV genome. The genome also contains numbered genes from the Unique Short, US, segment, with both segments flanked by a series of repeated sequences). The other product (GenBank accession number U09213) did not match to the HCMV genome, or at the time to any other viral, cellular or bacterial sequence. A description of the initial culturing and growth characteristics of the virus, along with an analysis of the sequence data was published in 1994. 6

DNA was isolated from the prototype culture using ultracentrifugation to isolate viral particles. In the first series of experiments, the DNA was cut with the enzyme EcoRI. In a second experiment agarose gel banded purified DNA was cut with the enzyme SacI. The cut DNA was cloned into plasmids and many of the clones were sequenced. As each sequence became available for analysis, it was compared with the known complete sequence of HCMV, and the available sequence data of all other viruses, including CMV of various animals.

The sequence of some of the clones corresponded to regions for which the sequence data were available on both rhesus cytomegalovirus (RhCMV) and African green monkey simian cytomegalovirus (SCMV). Sequence comparison led to the unequivocal conclusion that the virus had originated from a SCMV. 12 By comparing PCR results using primers sets directed against sequences of other parts of the stealth virus and other regions of SCMV, it was clear that the stealth virus had diverged significantly from SCMV. It lacked several genes corresponding to known regions of HCMV that would also be expected in SCMV. An update of these data is that also includes comparison with baboon CMV (BaCMV) is provided in Table 1.

Much of the sequence data were included in a series of peer-reviewed publications, 13-18 and submitted to GenBank. Most noteworthy were the findings indicating: i) Apparent lack of genes coding the virus components known to be targeted by the majority of cytotoxic T lymphocytes reactive with CMV infected cells. ii) Genetic instability and fragmentation of the virus genome. iii) Incorporation of cellular sequences, including a potential cancer causing gene, into the stealth virus. iv) Increased gene-copy number, when compared to HCMV, of the US28 gene. This gene codes a cell receptor molecule that binds to a class of cell activators, termed chemokines, which belong to a group of cell signaling molecules called cytokines. More interestingly, the US28 gene product provides a cell entry molecule for HIV. v) Presence of unusual bacteria-derived sequences in the culture. Several of the sequences matched very closely (but not identically) to alpha-proteobacteria, especially Brucella. The homology extended to the spirochete Borellia (the cause of classic acute Lyme disease). Other sequences matched more closely to mycoplasma (implicated in CFS and Gulf was syndrome), Streptococcus (implicated in childhood obsessive compulsive disorders and PANDAS syndromes) and to other distinct types of bacteria. The finding of these sequences raised the distinct possibility that stealth virus infected patients could be mistakenly identified as being infected with these various types of bacteria. vi) The second PCR product isolated from the original stealth virus culture was shown to unequivocally correspond to the UL19 region of SCMV. vii) A closely related sequence was identified in a PCR product isolated from the patient with the bi-polar illness. 19

Disease Outbreaks

The infectious nature of stealth-adapted viruses has been repeatedly suggested by individuals reporting their illness beginning shortly after a sexual and even non-sexual encounter with a symptomatic individual. Some patients have linked their illness to having received a blood transfusion or gamma globulin injection. Distinct community wide outbreaks have also come to the attention of various clinicians. One such outbreak occurred in the Mohave Valley region of Western Arizona in the spring of 1996. 20 Over 100 patients presented during a three month period with an acute gastrointestinal syndrome, followed by persisting fatigue, cognitive dysfunction and personality changes. Since then the community has experienced widespread chronic illnesses, including heightened allergies, chemical sensitivities especially to pesticides, higher than expected learning disorders among its children, and a high incidence of both depression and psychosis. Stealth virus cultures from these patients were consistently positive and distinguishable from control cultures in both routine and in double blinded independent studies. The clinician overseeing many of these cases encountered at least 10 fatalities among symptomatic, virus-culture positive, middle-aged individuals. He also referred a child with a slowly evolving behavioral and learning disorder. 21 An organic component was finally recognized by his parents, both of whom were physicians. In spite of an essentially normal clinical neurological examination, an MRI showed extensive sub-cortical T1 and T2 abnormalities. A brain biopsy showed the characteristic foamy vacuolating changes seen in other patients and in stealth virus inoculated cats. Mitochondria damage and intracellular inclusions were also evident. Both the mother and her son were culture positive. The child showed a significant response to ganciclovir (an anti-herpesvirus drug). The infecting virus could not be identified as a derivative of SCMV using a PCR based assay. This virus was also deposited with the American Type Culture Collection (Accession number VR-2568). Other community wide outbreaks of stealth-adapted viruses have been partially investigated and include Joelton , TN and Peoria , IL . As noted above, illnesses occurring within families also attest to the potential infectious nature of stealth-adapted viruses. Positive cultures and PCR based assays have also frequently been obtained from cancer patients including patients with salivary gland tumors, 22 breast cancers and multiple myeloma.

Stealth Virus Inhibitor

Experience gained with the culturing of stealth-adapted viruses indicated the need to frequently replace the tissue culture medium. It was presumed that some factor or factors were accumulating in the cultures that were preventing the progression of the CPE. Insight into the inhibitory substances has come from recent studies on the aggregated pigmented intracellular materials seen in the virus cultures 23 and in the vacuolated cells present in various brain biopsies. 24 These materials are potentially providing an alternative energy source for the infected cells. The mineral containing pigments appear capable of converting various forms of physical energies to chemical energy. 23 They have been designated alternative cellular energy pigments (ACE)-pigments.

Summary and Conclusions

The following conclusions can be drawn from the work. i) Atypically structured, non-inflammation inducing (stealth-adapted) cytopathic viruses definitely exist. ii) Some of these viruses were derived from SCMV and have presumably entered the human population from SCMV contaminated polio vaccines. iii) Stealth-adapted viruses can be regularly cultured from patients with complex multi-system illnesses, including various cancers. iv) Stealth-adaptation is considered to be a generic process that can involve many types of cytopathic viruses. It presumably occurs through the loss of genes coding for major antigens normally targeted by the cellular immune system. v) Tissue culture provides the best means to screen for stealth-adapted viruses. Viral cultures can also provide useful insights into pathology, including formation of lipids, and energy transducing pigments. vi) The production of lipids and pigmented material is viewed as a reparative process helping to maintain cell viability. This is based on the marked reduction in the intensity of the cytopathic effect if the culture medium is not frequently replaced. vii) Bacterial and cell derived genes are in the SCMV-derived stealth virus culture. This finding indicates the potential intermixing of cellular, viral and bacterial genes in the creation of new highly pathogenic microorganisms. viii) Stealth viruses are found in certain cancer patients, most of whom have symptoms of an underlying neuropsychiatric illness. The prospect of bacteria transmitting cancer causing viruses is a very serious and urgent public health concern. ix) Bacterial genes can help explain partial and inconsistent serological and PCR diagnostic findings for different bacteria in stealth virus infected individuals. x) The present studies provide support for an infectious process as a major contributing factor in the etiology of many chronic illnesses including cancer.

24. Martin WJ. Complex intracellular inclusions in the brain of a child with a stealth

virus encephalopathy. Exp Mol Path (in press).

Table 1. SCMV Related Genes in the Cultured Stealth-Adapted Virus

CMV-Related Matching of Stealth Virus Sequences to

Contiguous SCMV BaCMV RhCMV HCMV

Sequences nucleotide matching (Expect value)

Genes Length

UL 14 1,458

UL 19-48 38,262

UL 48-54 8,407 UL50 571/598 422/465 384/453 159/185

UL 56 767 (0.0) (e-180) (e-95) (e-38)

UL 57 + ori 2,748 UL57 1226/1246 499/577 1011/1265 370/492

UL 61-69 4,459 (0.0) (0.0) (0.0) (e-62)

UL 70 1,729

UL 71-76 6,328

UL 77-78 1,884

UL 84-104 25,023 UL93 630/656 575/647 368/406 266/317

UL 104-105 1,464 (0.0) (0.0) (e-137) (e-57)

UL 111-112 1,955 UL111 760/807 709/901 74/89 75/89

UL 115-132 8,628 (0.0) (e-171) (e-7) (e-15)

UL 141-144 5,820

US18

US 20-29* 16,011

US 30-32 3,978

Analysis based on >300 clones of DNA of stealth virus culture. There is good evidence that the virus has a fragmented, unstable genome. Note apparent absence of UL 83 and UL 55. The UL 123 showed significant mutations. The products of these three genes would ordinarily provide the major antigens recognized by anti-CMV cytotoxic T cells. The nucleotide homology data establish that this particular stealth-adapted virus was derived from SCMV.